Clindamycin

A stock solution of nisin (1000 μg/mL) was obtained by dissolving 1 g of nisin powder (2.5% purity Sigma-Aldrich, USA) in 25 mL of HCl (0.02 M) (Merck, Germany), filtered using a 0.22 μm cellulose acetate membrane filter and stored at 4°C. The stock solution was then diluted with sterile water to a concentration of 45 μg/mL.
A guar gum gel 1.5% (w/v) was prepared by dissolving 0.6 g of guar gum (Sigma-Aldrich, USA) in 40 mL of sterile distilled water and heat sterilized by autoclave. The solution of nisin was incorporated within the guar gum gel in a proportion of 1:1, obtaining a final 0.75% (w/v) biogel with 22.5 μg/mL of nisin.
Regarding antibiotics solutions, 6.6, 4.76 and 10.62 mg of Clindamycin (Cayman, USA), Gentamicin (PanReac AppliChem, USA) and Vancomycin (PanReac AppliChem, USA), respectively, were dissolved in 10 mL of sterile water and filtered through a 0.22 μm cellulose acetate membrane filter. Stock solutions were kept frozen at -80°C and diluted to the final concentrations of 0.033 μg/mL for Clindamycin, 0.238 μg/mL for Gentamicin and 0.531 μg/mL for Vancomycin, prior to utilization.

Stock solutions of gentamicin (ITW Reagents, Monza, Italy) were prepared at 4760 µg/mL, clindamycin (Cayman Chemical, Michigan, MI, USA) at 660 µg/mL, and vancomycin (ITW Reagents, Monza, Italy) at 1062 µg/mL, according to the manufacturer’s specifications, and kept at −80 °C. Antibiotic working solutions were used at their minimum inhibitory concentration (MIC) values (gentamicin, 0.238 µg/mL; clindamycin, 0.033 µg/mL; and vancomycin, 0.531 µg/mL), as determined in a previous study [65 (link)].
Nisin A (1000 UI/mg, 2.5% purity) (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 0.02 M HCl (Merck, Darmstadt, Germany), yielding a 1000 µg/mL stock solution, and pexiganan (>95% purity; Innovagen, Lund, Sweden) was dissolved in deionized sterile water, yielding a 2048 µg/mL stock solution. These solutions were then filtered through a 0.22 µm filter (Millipore Corporation, Billerica, MA, USA) and kept at 4 °C. To prepare the dual-AMP biogel, a pexiganan solution at 256 µg/mL was enriched with Nisin At 125 µg/mL, corresponding to their minimum biofilm eradication concentration (MBEC) values, as determined previously [25 (link)]. 0.75 g of guar gum (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 50 mL of sterile distilled water, yielding a 1.5% (w/v) gel, which was sterilized by autoclave. Antibiotics and AMPs were mixed into the gel in a 1:1 ratio.